(1) Field of The Invention
This invention relates to the fields of detection of substances in biological samples and, more particularly to the localization and the quantitative analysis of substances in the same biological sample.
(2) Description of The Related Art
Diagnostic laboratories have used antibodies, lectins, nucleotide probes and the like for many years to localize target substances in cells and tissues. The binding of such probes to the target substance is typically detected microscopically by the use of direct labeled probes such as fluorophores, enzyme conjugates, gold particles and the like or by conjugated "secondary" probes that recognize the "primary" probe. This basic protocol continues to be used in numerous clinical and research laboratories. Recent advances in detection systems have improved the sensitivity and resolution of the probe localization and include such methods as immunogold with silver intensification, peroxidase-anti-peroxidase, avidin-biotin complex, and tyramide signal amplification. Although these methods provide excellent spatial resolution of antigen distribution, they do not provide any direct quantitative information about the target substance. One method for obtaining such quantitative information has been by the use of image analysis, however, this approach requires a significant amount of time and expense.
Numerous techniques exist for the quantitation of substances in cells and tissues including Western, Southern and Northern blots, radioimmunoassays, ELISA assays and dot blot assays. While these techniques can provide useful quantitative information, they typically provide no information at all about the localization of the target substance within the cells or tissues. Furthermore, the methods often require tissue extraction and/or disruption which precludes localization of the target substance. Nevertheless, it is often essential in the clinical laboratory setting to obtain information on both the distribution of a substance within a cell or tissue as well as the quantitative amount of the substance present in the tissue.
Although some approaches have reported on attempts to obtain information in addition to the histochemical localization or in addition to the quantitative amount of the substance present, few reports have been published on approaches other than image analysis achieving both localization and quantitation of a substance in a biological sample. In some studies, the quantitation of a substance present in a cell suspension has been coupled with a determination of the number of positive cells, i.e. a cell count (for example see, Makler et al, Transfusion21:303-312, 1981). Such studies, however, provide no information on the localization of a target substance within the cell suspension.
In another report, quantitative detection has been performed that uses an immunohistochemical technique used earlier for antigen localization. (Leuven et al., J Immunol Meth23:109-116, 1978). The method reported in this study, however, disrupted the cells prior to quantitative determination of the antigen. Thus, in both the Leuven et al. and Makler et al. methods, to perform both quantitation and localization, duplicate preparations were indicated to be required.
Another group reported on an assay system in which both quantitation and localization was determined in the same sample (U.S. Pat. No. 4,487,830). This method utilized a fluorescent antibody to detect the localization of the target substance and an enzyme-linked antibody to quantitate the presence of the target substance or, alternatively, a fluorescent antibody conjugated to an enzyme. However, fluorescent antibodies provide minimal signal amplification and require microscopes with epifluorescence attachments for visualization of signal which are not routinely available in clinical laboratories.
In another report a probe labeled with a chemiluminescent or bioluminescent substance was used that reacted with other substrate molecules to produce light (U.S. Pat. No. 4,478,817). Localization was by visual inspection and quantitation could be obtained from the total of light emitted from the sample using, for example a photo multiplier tube. Such quantitation is difficult to perform and time consuming and requires equipment not routinely found in clinical laboratories. Furthermore, none of the above cited references describe procedures for multi-probe detection. Thus, variance in tissue or cell amounts can not be determined on single samples using these techniques.
Thus it would be desirable to have a simple and inexpensive method for detecting both the localization and quantitation of a substance in cells or tissues that can be used in virtually any clinical laboratory and that does not require fluorescent microscopy or image analysis.